A wide range of infectious agents, including infectious proteins, spore forming bacteria, vegetative bacteria, fungus and viruses have major impacts in medical settings. The process to remove infectious organisms or render them non-infectious from medical equipment makes use of a wide range of sterilization devices or equipment and disinfection devices and processes. The CDC lists examples of infectious agents and microorganisms by resistance to standard disinfection and sterilization processes. See Table 1 below from CDC's Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008.
Some infectious agents, such as the HIV virus, may be easy to remove from medical equipment. Many infectious agents, including vegetative bacteria, are moderately difficult to eliminate. Other infectious agents, such as prions, can only be destroyed by extremely harsh conditions that damage and/or destroy modern medical equipment. Failure to eliminate infectious agents from medical equipment before use can put patients at extreme risk of injury and death.
TABLE 1Decreasing order of resistance of infectious agentsand microorganisms to disinfection and sterilization.Agent CategoryExample Organisms or DiseasesPrionsCreutfeldt-Jakob DiseaseBacterial sporesBacillius atrophaeusCoccidicaCryptosporidiumMycobacteriaM. tuberculosis, M. terraeNonlipid or small virusespolio, coxsackieFungiAspergillus, CandidaVegetative bacteriaS. aureus, P. aeruginosaLipid of medium-sized virusesHIV, herpes, hepatitis B
Some conventional methods to determine if sterilization equipment functions effectively may rely on FDA approved Biologic Indicator process (BI strips) in a multi-step process. This widely accepted conventional process starts with filter papers infused with a defined number of bacterial spores (BI strips). The BI strips are subjected to a standard cycle by the sterilization equipment or device, e.g., an ethylene oxide (EtO) sterilization, radiation, or steam sterilization equipment which is being qualified. After the sterilization process is completed, the treated strips are then placed in a defined bacterial media for growth, frequently for days to weeks. If no growth is seen after the defined period, the sterilization process by the medical equipment being certified is declared a success. Together this combination of supplies and techniques is the approved process to qualify sterilization equipment in positive or negative process. If there is growth, the sterilization equipment fails and if there is no growth the sterilization equipment passes.
The conventional biologic indicator tests may use one of three different species of bacteria. The standard species used to test the effectiveness of ethylene oxide (EtO) sterilization is B. atrophaeus. To test the effectiveness of gamma radiation sterilization, the bacteria species used is B. pumilis. To test the effectiveness of steam sterilization the bacteria species used is G. stearothermophilus. However, the three species that are used to qualify sterilization capacity of equipment are not bacteria that commonly cause disease in humans. Instead, the species are surrogate species, strains of soil bacteria that form high persistent spores. They are used instead of medically relevant infectious agents, because, inter alia, the spores of the bacteria are extremely difficult to damage such that they can no longer replicate, and if for some reason a health care worker or patient accidently comes in contact with the spores through use or on improperly cleaned equipment, there is very little chance that the human will become ill. As spores from the surrogate species are scientifically known to be more difficult to destroy than medically relevant species, such as Polio or S. aureus, e.g., Methicillin-resistant Staphyloccus aureaus (MRSA), when the sterilization equipment is qualified to destroy all spores on a BI test strip, the FDA accepts that the equipment is also able to destroy all organisms that rank lower for resistance to sterilization.
The conventional methods discussed above used to qualify effective sterilization, deimmunization, or disinfection may only measure the ability of the surrogate organisms to grow after sterilization treatment. However, such conventional methods do not indicate how the surrogate organisms are damaged and/or destroyed resulting in the absence of growth. Conventional surrogate testing methods also require the accurate production, storage, transport and handling of 10 thousand to 100 million pure bacteria spores, proper control of growth medias, extended period of growth of the specific spores and careful protection of all growth materials for environmental contamination to qualify if all the test surrogate organisms were completely eliminated. If any component of the process is not vigorously controlled, the sterilization qualification could give false positive or false negative results. False positive results will trigger extensive effort to unnecessarily repair sterilization equipment as well as the recall of days or weeks of sterilized medical equipment and the patients treated with such equipment. False negative results are worse because they will result in defective sterilization equipment being used and the resulting contaminated medical equipment endangering patients.
For other infectious organisms, such as members of the bacterial genera Clostridium, Staphylococcus or fungal genera Trichophyton or Candida, and the like, specific tests for each genera may be based on similar fundamentals. See Table 2 below for a list of common bacteria and fungus genera and species having an impact on human medicine.
TABLE 2Common bacteria and fungus genera and specieshaving an impact on human medicine.Health Care Application - Problempathogen or BL1/sterilizationGenusExample Speciestesting organism.BacillusBacillus subtilisResearch organism, used as an“indicator organism” duringdisinfection testing. BL1.Bacillus atrophaeusUsed as an “indicator organism”during gas (EtO) sterilizationprocedure. BL1.Bacillus pumilisUsed as an “indicator organism”during radiation sterilizationprocedure. BL1.Geobacillus stearothermophilusUsed as an “indicator organism”(formerly B. stearothermophilus)during steam sterilizationprocedure. BL1.Bacillus anthracisCauses anthrax.Bacillus cereusCauses food poisoning similar tothat caused by Staphylococcus.ClostridiumClostridium sporogenesUsed as a surrogate forC. botulinum when testing theefficacy of commercialsterilization.Clostridium tetaniCauses tetanus.Clostridium botulinumCauses botulism poisoning.Clostridium perfringensCauses gas gangreneClostridium difficileCauses C. dif GI infection.Clostridium novyiCauses a wide range of human andanimal infections depending ontype.MycobacteriumMycobacterium tuberculosisMajor cause of humantuberculosis.Mycobacterium africanumSlow growing form oftuberculosis.Mycobacterium capraeMore rare form of humantuberculosis.Mycobacterium kansasiiChronic human pulmonary diseaseresembling tuberculosis(involvement of the upper lobe).Mycobacterium ulceransInfects the skin and subcutaneoustissues, giving rise to indolentnonulcerated and ulcerated lesions.Mycobacterium interjectumChronic lymphadenitisMycobacterium lepraeCauses leprosyMycobacterium lepromatosisCauses leprosyMycobacterium terraeCauses serious skin infections thatare relatively resistant to antibiotictherapy.Used to study effectiveness ofdisinfection processes for reusablemedical instruments.Mycobacterium gastriCasual resident of humanstomachs, but not considered anetiologic agent of disease. (BL1).StaphylococcusStaphylococcus aureusCauses a variety of infections inthe body, including boils, cellulitis,abscesses, wound infections, toxicshock syndrome, pneumonia, andfood poisoning.Substrain - Methicillin-resistantStaphylococcus aureus (MRSA).Sub-strain - Vancomycin-resistantStaphylococcus aureus (VRSA) -acquired gene from VRE.Staphylococcus capitisAssociated with prosthetic valveendocarditis, forms biofilms.Staphylococcus epidermidisHospital-acquired concern as itforms biofilms catheters or othersurgical implants.Staphylococcus haemolyticusSecond-most frequently isolatedhospital-acquired Infection, oftenassociated with the insertion ofmedical devices; highly antibiotic-resistant phenotype and able toform biofilms.Staphylococcus lugdunensisWide variety of infectionsincluding cardiovascularinfections, osteomyelitis andprosthetic/native joints infections,skin and soft-tissue infection,central nervous infections,peritonitis, endocephalitis, andurinary tract infections.Staphylococcus saccharolyticusMay cause of infectiveendocarditis.Staphylococcus saprophyticusCommon cause of community-acquired urinary tract infections.Staphylococcus auricularisOccasionally can be involved withhuman skin infections.SalmonellaSalmonella entericaCauses food poisoning.EnterococcusEnterococcus faecalisCan cause endocarditis andsepticemia, urinary tract infections,meningitis, and other infections.Substrain - Vancomycin-resistantEnterococcus (VRE).Enterococcus faeciumNeonatal meningitis orendocarditis.Substrain - Vancomycin-resistantEnterococcus (VRE).Enterococcus gallinarumKnown to cause outbreaks andspread in hospitals.Enterococcus hiraeEndocarditis and septicemia inhumans.Enterococcus malodoratusFrequently the cause of hospital-acquired noscomial infections,bloodstream infections, and urinarytract infections.EscherichiaEscherichia coliSome serotypes can cause seriousfood poisoning in their hosts.Substrain K-12 strain commonlyused in recombinant DNA work(BL1).Substrain O157:H7 causes seriousillness or death in the elderly, thevery young, or theimmunocompromised.Substrain O104:H4, can triggermajor cause of foodborne illnessand lead to hemolytic-uremicsyndrome (HUS).Escherichia fergusoniiKnown to infect open wounds andmay also cause bacteraemia orurinary tract infections; highlyresistant to the antibiotic ampicillinand some also resistant togentamicin and chloramphenicol.HelicobacterHelicobacter pyloriCause gastritis and ulcers.Helicobacter hepaticusMay be associated with Crohn'sdisease and ulcerative colitis.Helicobacter bilisMay be associated with Crohn'sdisease and ulcerative colitis.Helicobacter ganmaniMay be associated with Crohn'sdisease and ulcerative colitis.KlebsiellaKlebsiella pneumoniaeCauses pneumonia, urinary tractinfections, septicemia, meningitis,diarrhea, and soft tissue infections;naturally resistant to manyantibiotics.Substrain - CREs - carbapenem-resistant Klebsiella pneumoniae(CRKP).Klebsiella oxytocaCause colitis and sepsis.NeisseriaNeisseria gonorrhoeaeCauses Gonorrhea.Neisseria meningitidisCauses meningitis.PseudomonasPseudomonas aeruginosaA multidrug resistant pathogenassociated with hospital-acquiredinfections such as ventilator-associated pneumonia and varioussepsis syndromes. Common in CFpatients.Pseudomonas mendocinaOccasionally causes hospital-acquired infections, such asinfective endocarditis andspondylodiscitis.Pseudomonas fluorescensProduces enzymes that cause milkto spoil and occasionally infectsimmunocompromised patients.Pseudomonas putidaUsed in bioremediation, or the useof microorganisms to biodegradeoil.TrichophytonTrichophyton rubrumMost common cause of athlete's(Fungus)foot, fungal infection of nail, jockitch, and ringworm.Trichophyton tonsuransCauses ringworm infection of thescalp.Trichophyton interdigitaleOne of three common fungi whichcause ringworm.Trichophyton mentagrophytesCauses tinea infections includingathlete's foot, ringworm, jock itch,and similar infections of the nail,beard, skin and scalp.Trichophyton concentricumAssociated with the skin infectiontinea imbricate.CandidaCandida albicansDimorphic fungus that grows both(Fungus)as yeast and filamentous cells;Responsible for 50-90% of allcases of candidiasis in human.Important causes of morbidity andmortality in immunocompromisedpatients. Biofilms may form on thesurface of implantable medicaldevices. Cause of 85-95% ofvaginal infections cases areresponsible for physician officevisits every year.Candida dubliniensisA fungal opportunistic pathogenoriginally isolated from AIDSpatients. It is also occasionallyisolated from immunocompetentindividuals.Candida tropicalisCommon pathogen in neutropaenichosts; research suggests thatC. tropicalis, workingsynergistically withEscherichia coli andSerratia marcescens. May cause orcontribute to Crohn's diseaseCandida aurisCauses candidiasis in humans;often acquired in the hospital whenhuman immune systems areweakened; causes fungemia,yielding candidemia (systemiccandidiasis); attracted clinicalattention because of multidrugresistance.
Examples of standardized methods for sterilization or disinfection (A) and standardized testing methods protocols (B) used to determine the effectiveness of sterilization or disinfection, as shown in Table 3 below:
TABLE 3Approved Methods of Sterilization or Disinfectionand Qualifying Test Protocols(A) Standard Methods for Preparing Healthcare EquipmentDisinfectionSterilizationAlcoholSteam SterilizationChlorine and Chlorine CompoundsFlash SterilizationFormaldehydeEthylene Oxide “Gas”SterilizationGlutaraldehydeHydrogen Peroxide Gas PlasmaHydrogen PeroxidePeracetic Acid SterilizationIodophorsIonizing RadiationOrtho-phthalaldehydeDry-Heat SterilizersPeracetic AcidLiquid ChemicalsPeracetic Acid and Hydrogen PeroxidePerformic AcidPhenolicsFiltrationQuaternary Ammonium CompoundsMicrowaveRadiationGlass Bead “Sterilizer”PasteurizationVaporized Hydrogen PeroxideFlushing- and Washer-DisinfectorsOzoneFormaldehydeGaseous Chlorine DioxideVaporized Peracetic AcidInfrared radiation(B) Standard Test to Qualtify Healthcare EquipmentTest NameExample Test SpeciesBI (ethylene oxide (EtO) sterilization)B. atrophaeusBI (gamma radiation sterilization)B. pumilisBI (steam sterilization)G. stearothermophilusAOAC Sporicidal Efficacy Test MethodClostridium sporogenesBacillus subtilisAOAC Tuberculosis Rate of KillMycobacterium terraeAOAC Use Dilution TestPseudomonas aeruginosaStaphylococcus aureusSalmonella entericaAOAC Fungicidal Efficacy Test MethodTrichophyton mentagrophytes
For each standard test protocol, a define number of organisms are placed on a carrier, such as tube, filter paper, or coated on and in a test solid surface. The specific organisms may be a particular infectious species or could be a surrogate species of the same genus that is closely related to the infectious species. In all cases, the species, carrier and growth conditions are defined by the FDA and/or the Association of Analytical Communities (AOAC) protocol. Following treatment with sterilization or disinfection equipment, the carrier with the specific species sample is placed into ideal growing conditions for the particular test species. After a required period in culture, usually 2 to 30 days, the culture is monitored. If no growth is observed, the sterilization or disinfection equipment is declared to be operating within required parameters.
In addition to enabling growth and infectivity, protein components of infectious organisms could trigger severe immunogenic or allergic reactions in susceptible individuals even at very low level. Examples include mold proteins that are able to trigger severe allergic reactions even if the mold has been rendered no longer able to grow. Immunogenic proteins can also occur in food such as gliadin, a highly immunogenic protein component of the seed storage protein gluten in wheat and related grains. Gliadin can trigger reactions in most individuals suffering from Crohn's disease. It is critical that immunogenic proteins are completely removed from any equipment that will be used in conjunction with susceptible individuals.
A wide range of pathogenic organisms use a multicopper oxidase with 3 cupredoxin superfamily domains for growth and survival. As disclosed herein, the loci suf I that contains a critical protein that confers different functions depending on the genus (bacteria or fungus) and this critical protein can be targeted. Depending on the genus, the suf I loci encoded protein can have different names. The functions of the protein encoded by suf I include cell division (FtsP), formation of spore coat proteins (CotA), chromosome partitioning, inorganic ion transport, and metabolism and cell wall, membrane, and envelope formation. As the protein product of the suf I loci are absolutely critical for the survival of the spores (in spore forming bacteria) and/or growth (all bacteria and fungus), if the protein product of the suf I loci is irreversibly fragmented into short polypeptides and amino acids, the bacteria or fungus cannot survive. Additionally, it is likely that a sterilization method that clearly demonstrates fragmentation of the protein product of the suf I loci would also fragment other proteins in the bacterium or fungus. Bacteria and fungus can be divided into distinct genus each containing multiple species. Many species also have subspecies that carry unique characteristics include multi-drug resistance. In human health situations, certain bacteria and fungus species and subspecies are of major concern because they are capable of causing disease. Related species may be used in medical research, e.g., E. coli KI2, or as indicator species for qualification of sterilization, e.g., B. atrophaeus used to qualify gas sterilization. See Table 2 above for a list of common bacteria and fungus genera and species with impact on human medicine.
Prions are a unique category of a transmissible infections agent that comprised only of protein, without DNA or RNA. Prions can cause a wide range of neurodegenerative diseases known as transmissible spongiform encephalopathies (TSE) or prion diseases including the new variant Creutzfeldt-Jakob disease (nvCJD). See Table 4 below. Infectious prions are in fact an abnormally folded brain protein. This brain protein (Protease resistant Proteins, PrP) can be folded into two different structural (tertiary) forms, the normal brain protein, PrPc, and the abnormal, disease triggering form, PrPsc. The disease triggering form, PrPsc, is found in high quantity in the brain of infected humans and animals and can be transferred to a new host with the transfer of infected material. Once in the new host, the abnormally folded protein (PrPsc) causes disease symptoms by promoting the unfolding of the normal host protein (PrPc) and refolding into the disease causing form (PrPsc). PrP proteins can also be partially cleave and still retain their infectious characteristics. Full length mature PrP protein (both PrPc and PrPsc) is 209 amino acids long. Limited proteolysis of PrPsc will cleave amino acids from the amino terminus resulting in another infectious protein form PrP 27-30 that is approximately 142 amino acids long. Additional cleavage that significantly reduces the 142 amino acid long PrP 27-30 is needed to render the PrP protein irreversibly non-infectious. Although most infectious agents can be permanently rendered non-infectious by heat or steam, these methods are not sufficient to eliminate infectious prions from medical equipment.
TABLE 4Example of Prion Diseases in Different Species and Potential Origin of the Infectious Protein.DiseaseSpeciesPotential OriginDiseaseSpeciesPotential OriginCreutzfeldt-JakobHumanInheritedScrapieSheep andInherited/disease (CJD)GoatenvironmentalNew VariantHumanConsumption,Bovine SpongiformCattleConsumptionCreutzfeldt-JakobMedicalEncephalopathydisease (CJD)Contamination(BSE)Fatal FamilialHumanInheritedTransmissible MinkMinkEnvironmentalInsomnia (FFI)Encephalopathy(TME)Gerstmann-StrausslerHumanInheritedChronic WastingMule DeerEnvironmentaldisease (GSD)Disease (CWD)and ElkHuntington disease-HumanInheritedFeline SpongiformCatsConsumptionlike type 1 (HDL1)Encephalopathy(FSE)KuruHumanConsumption ofExotic UngulateNyala andEnvironmentalHuman BrainsEncephalopathyGreater(EUE)Kudu
As discussed above, prions are abnormally folded protease resistant proteins (PrPsc) that cause disease symptoms by promoting the unfolding of normal proteins (PrPc) and refolding into the disease causing protein form (PrPsc). As the level of the PrPsc rises in the patient's brain, symptoms of progressive dementia, myoclonic seizures, abnormalities of high cortical function, cerebellar and corticospinal disturbances develop. The period between infection and development of disease can extend for years to decades. The duration of disease symptoms is variable but is typically 8 to 18 months.
Once prion proteins fold into the infectious form (PrPsc), they are extremely difficult to render non-infectious. Conventional methods to sterilize medical equipment contaminated with prions, such as high heat to promote loss of function of other protein types by triggering loss of tertiary structure, are ineffective because unlike most proteins, the denatured prion proteins, both infectious and non-infectious, will spontaneously refold by themselves back to their pre-treatment forms. In some cases, conventional methods may actually result in refolded into infectious form promoting the conversion of the non-infection prion protein into the infectious prion protein.
To render infectious proteins such as prions irreversibly non-infectious, all infectious proteins must be fragmented into small polypeptides, amino acids or components. The only currently approved conventional method for this process Is harsh treatment of medical equipment and supplies with caustic soda, an extremely harsh process that frequently damages and/or destroys medical equipment
Determining whether or not an infectious prion (PrPsc) sample has been permanently destroyed can be extremely difficult and time consuming. Conventional methods for determining whether an infectious prion has been permanently destroyed require that after attempted deactivation, the PrPsc sample is injected into a matched susceptible animal that is then followed for an extended time to see if the animal develops disease. In larger animals, the process can take years, but even in a small animal such as a mouse, the test can take months. As there is a potential for inter-animal variation and poor test accuracy, a large animal test pool is required to obtain relatively accurate results.
Immunogens may include a wide range of molecules including proteins that can trigger dramatic immunologic responses in susceptible individuals. The responses can trigger serious allergic reactions on the skin (e.g., poison ivy rash), in the gut (e.g., triggering a flare-up in Crohn's disease), in the lung (e.g., asthma) or a systemic response (e.g., anaphylaxis). Protein immunogens are a special class of immunogens produced by a wide range of bacteria, fungus (e.g. mold) or plants and can be difficult to destroy. An example of a common plant immunogen is gluten. Common grains such as various strains of wheat, farro, rye and spelt are derived from wild and domesticated grains of the Triticum, Aegilops and Secale genera. Common to all these species is the seed storage protein complex called gluten. When seeds are ground into flour, the gluten protein complex gives bread dough its elastic quality and bread its spongy texture. Unfortunately gluten is comprised of several proteins including Gliadin (also called Prolamin) which triggers severe T cell attack on the gut of patients with the autoimmune disease Celiac disease (CD). Gliadins can be typed as α, γ, and ω with a small protease resistant fragment (p57-73) of α-gliadins triggering the most severe destructive T cell response. As a results CD patients must not only avoid products containing gluten, but also need to be extremely careful to avoid small amounts of residual α-gliadin that may contaminate food preparation utensils.
Protease resistant proteins like α-gliadin are resistant to destruction so it is critical that devices and methods used to destroy them and other immunogens (also called allergens) can be easily checked to ensure they are operating at peak efficiency. If not, residual allergens can trigger life threatening responses in sensitive patients. The process of removing immunogens by deimmunization methods or devices is called deimmunization. The ability to test for the destruction of different immunogens on surfaces is not standardized. Usually affected patients are subjected to skin test regiments to determine their individual reactions to different candidate immunogens/allergens. The patient is then advised to avoid all immunogen contact and discard any materials potential contaminated with the specific immunogen or allergen. In cooking and manufacturing situations, extreme care must be taken to avoid potential cross contamination to the point that food packaging labels frequently carry warning labels about the potential issues.
Thus, a method is needed to determine irreversible destruction of proteins critical for the growth of infectious organisms, immunogenic proteins, and/or infectious proteins (e.g., prions) and thus rapidly and accurately determines the effectiveness of sterilization, deimmunization, and/or disinfection of equipment or supplies by a device. With such a method for rapidly determining effective sterilization, deimmunization, and/or disinfection, medical personnel and patients can have confidence that the medical equipment used for patient treatment is not contaminated with potentially lethal or immunogenic proteins. Without such a method, medical personnel may believe they are using properly sterilized equipment and then later discover that they have accidentally exposed their patients to lethal infections and harmful immune reactions